Endochondral ossification is a very complex process and a great deal of research has determined that proteoglycans (PCs) have a critical role in cartilage calcification during this process. Previous work has demonstrated that PG aggregate size is a key regulator of crystal size and proliferation in calcified cartilage. Additionally, several knockout animal models have suggested that PG synthesis, structure, and degradation are intimately related to cartilage mineralization in the process of endochondral ossification. PG modification also influences the activity of surrounding cytokines, such as phosphoproteins. However, the mechanism by which PG morphology is regulated during cartilage calcification is still unknown. Thus we hypothesize that aggrecan modification is required for physiologic cartilage calcification to occur and that a delicate balance exists between proteoglycan morphology and protein activity ultimately resulting in cartilage calcification. To test these hypotheses, we propose to use a mouse mesenchymal cell line previously demonstrated to recapitulate the process of endochondral ossification in micromass culture. Our Specific Aims are to: 1) To determine the mechanism of aggrecan turnover within the growth plate. During different stages of chondrocyte maturation, the micromass system will be probed with monoclonal antibodies developed for specific neoepitopes that result from cleavage of aggrecan by MMPs, ADAMTSs, and cysteine proteases. 2) To test the hypothesis that aggrecan modification is required for physiologic cartilage calcification to occur using a combination of immune-blocking and RNAi knock down experiments for aggrecanolytic proteinases. 3) To determine the relationship between the degree and type of aggrecan sulfation and cartilage calcification. Micromass cultures will be treated with sodium chlorate (NaCIO3) to inhibit glycosaminoglycan sulfation of aggrecan, chondroitinase to release chondroitin sulfate (CS), or keratanase to release keratan sulfate (KS) chains from the matrix. The effects of these treatment groups on aggrecanolytic proteinase and phosphoprotein activity will be assessed. Conditions in which cartilage calcification is aberrant affect both the new born and the elderly in terms of birth defects, growth deformities, and inherited abnormalities. To prevent and treat these conditions it is essential to understand physiologic cartilage calcification. The ultimate goal of these studies is to understand the mechanisms involved in physiologic cartilage calcification during endochondral bone growth using a cell culture model. Using this information, target genes or biomolecules may be identified so that clinical solutions that treat the functional disorders of cartilage tissue may be developed.